Purification And Characteristics Of An Aflatoxin-Degrading Enzyme Separated From Pseudomonas Stutzeri F4

Aflatoxin is a group of highly toxic and pathogenic secondary metabolite mainly produced by the fungus, such as Aspergillus flavus and Aspergillus parasiticus. It is well known that food and feed are often contaminated by aflatoxin, which causes big economic losses and threatens the health of human and animals. Much attention has been paid to the treatment of aflatoxin contaminant. Pseudomonas stutzeri F4with high degrading activity of AFB1was isolated in the early work. Based on it, this thesis focus on the production of AFB1-degrading enzyme (ADE) by F4strain. The fermentation conditions were investigated and optimized in order to improve the level of enzyme production, and ADE was separated, purified and characterized. The main results are as follows:1. The fermentation conditions of ADE produced by Pseudomonas stutzeri F4were investigated and optimized, the effects of inoculum age, inoculum size, initial pH, fermentation time and temperature on ADE activity were tested by the single factor method. Based on the single factor experiments, the fermentation conditions was analyzed and optimized by using response surface methodology in a central composite design involving17experiments of three variables at three levels. When inoculum size and initial pH were3%and6.5, the theoretical optimum fermentation condition was that the age of inoculum was10.5h, the fermentation time was42.5h and temperature was30.3℃, respectively. The ADE activity in the fermented broth reached to2769U under the optimized conditions, which increased29%to that before optimization.2. ADE was separated and purified from broken cells of F4. ADE crude enzyme solution was obtained by55%saturation of ammonium sulfate precipitation. Then it was purified by DEAE-Sepharose Fast Flow chromatography. And it was separated into three elution peaks and the third presented the enzyme activity while the highest enzyme relative activity was55%at the time of some144min. Then the solution obtained by DEAE-Sepharose Fast Flow chromatography was divided into two elution peaks by Superdex G-100chromatography. And the second (in the range of some920to1200min) presented the enzyme activity while the highest enzyme relative activity was45%at the time of some980min. A total123-fold purification of110mg original enzyme solution and0.5mg ADE with the tatal enzyme activity of 32×103U and the specific activity of64×103U/mg was acquired by using these purification methods. It proved the purification method was effective.3. The characteristics of the enzyme was studied based on the purified ADE. The apparent molecular mass of ADE was estimated to be about24kDa by SDS-PAGE. ADE displayed the highest degradation activity of AFB1at30℃and pH6.0. Cu2+exhibit strongly inhabitation and other metal ions (Zn2+, Mg2+, Mn2+and Fe3+) had little effect on ADE enzyme activity. Finally, the Km of ADE was determined using Lineweaver-Burk plot method, and it equaled5.61X10-5mol/L.